CN104758948B - The preparation method and application of multi-functional antineoplastic target diagnoses and treatment medicine based on gold nano star - Google Patents

Abstract

The present invention relates to the preparation method and application of multifunctional anti-tumor targeted diagnostic and therapeutic drugs based on gold nanostars. It can effectively solve the problems of existing anti-tumor drugs with large side effects, poor biocompatibility and poor targeting. The method is: first The gold nanostar carrier is structurally modified and reacted with an amino compound containing a thiol group to obtain an amino-modified gold nanostar solution; then the amino-modified gold nanostar is reacted with a hydrazine-bonded compound with an activated carboxyl group to undergo an amide reaction to generate a terminal-connected hydrazine bond-modified gold nanostar solution; then use its terminal hydrazine bond to chemically react with doxorubicin containing a carbonyl group to form a pH-sensitive hydrazone bond compound; finally, this compound is reacted with thioallylated NGR through thioallyl bond reaction, the body of the present invention has good physical and chemical stability, simple preparation process, rich sources of raw materials, low cost, few toxic and side effects, can effectively inhibit the proliferation of tumor cells, and is an innovation in anti-tumor targeted diagnostic and therapeutic drugs.

Description

Preparation method of multifunctional anti-tumor targeted diagnosis and treatment drug based on gold nanostars and applications

technical field

The invention relates to medicine, in particular to a preparation method and application of a multifunctional anti-tumor targeted diagnosis and treatment drug (system) based on gold nanostars.

Background technique

Nano-gold/silver uses metal salts of gold and silver as raw materials, and synthesizes nanoscale gold or silver with different properties by controlling experimental conditions. At present, different synthesis methods can be used to synthesize spherical, rod-shaped, star-shaped and other shapes, so that nano-gold/silver has different physical and chemical properties, and its size is generally 10-100 nm. They have the following advantages: (1) The preparation process is simple and the dispersibility is good; (2) The dielectric and photoelectric properties can be adjusted; (3) The surface of the particles can be effectively combined with biomolecules (such as antibodies, hormones, proteins, etc.); ( 4) Good biocompatibility and stability; (5) Surface-enhanced Raman scattering (SERS) properties; (6) Nano-gold can absorb near-infrared light well and convert it into heat or generate singlet states Oxygen is an excellent photothermal/photodynamic material; (7) it can be used as a carrier of antitumor drugs.

Radio frequency (Radiofrequency, RF) is an abbreviation of high-frequency alternating electromagnetic waves, which means electromagnetic frequencies that can radiate into space, and the frequency range is from 300KHz to 30GHz. The most commonly used frequency is 13.56MHz. Under the action of 13.56 MHz radio frequency, the temperature of the nano-gold material solution is 10-20° C. higher than that of the nano-gold material solution without radio frequency irradiation. Radiofrequency therapy is to heat and treat local tissues of the human body through radiofrequency electromagnetic fields. Its role is mainly to kill tumor cells or tissues through the biological effects of induction, electrolysis, and thermal effects on tissues.

As a new type of nanomaterial, gold nanostars consist of a core and sharp branch structures. Both the core structure and the tip branch structure have plasmon resonance absorption peaks in the near-infrared region, and can generate reactive oxygen species under radio frequency irradiation. Including singlet oxygen, superoxide anion, etc. The quantum yield rate of pure oxygen in gold nanostars is significantly higher than that of other representative photosensitizers (such as: porphyrins), and it is non-toxic to the human body. Therefore, it is expected to be used as a photodynamic therapy therapy. photosensitizer. Gold nanostars are highly reactive to generate reactive oxygen species under radiofrequency irradiation, and can exhibit obvious cytotoxicity, such as cleaving DNA in cells, inhibiting cell mitosis and growth, and inhibiting the activity of cellular proteolytic enzymes. It can be deduced that gold nanostars can be used for radiofrequency treatment of tumors.

Gold nanostars are prone to aggregation and agglomeration in the presence of high salt and some biomolecules (nucleic acid, protein, etc.), and their stability is poor. In order to improve the stability and biocompatibility of gold nanostars, we linked thiolated polyethylene glycol and polyetherimide to the surface of gold nanostars through the sulfur-gold bond formed by sulfhydryl and gold. Linking water-soluble polyethylene glycol with long circulation characteristics to the surface of gold nanostars can not only take advantage of the steric hindrance effect, improve the stability of gold nanostars, prolong the circulation time in vivo, and reduce toxicity, but also the polyethylene glycol carried Different side chain structures provide effective modification sites for the targeted modification of the surface of gold nanostars. In addition, mercapto-polyetherimide contains more protonated nitrogen atoms of multi-level ammonia, which can capture a large number of protons in acidic lysosomes, resulting in osmotic swelling and rupture of lysosomes, and then the endocytosed drugs Rapidly released into the cytoplasm to achieve lysosomal escape.

Studies have shown that the NGR motif can specifically target the aminopeptidase N (CD13) of tumor blood vessels, but has little effect on the aminopeptidase expressed in normal myeloid cells or epithelial cells. Therefore, in this study, NGR was used as a tumor targeting molecule, and the NGR polypeptide modified with cysteine was used to connect to the surface of gold nanostars through sulfur-gold bonds to achieve tumor targeting.

Doxorubicin is a broad-spectrum chemotherapeutic drug, which is commonly used clinically to treat malignant tumors such as breast cancer, children's solid tumors, soft tissue tumors, and nauseated lymphoma. Clinical studies in recent years have found that the resistance of tumor cells to doxorubicin and its potential cardiotoxicity limit its further application. Long-term use of doxorubicin can easily lead to myocardial damage and congestive heart failure. Clinical tumor pathology studies have found that when tumor tissue ischemia, abnormal acidification of tumor interstitial fluid and the fact that the pH is lower than normal tissue. Therefore, in view of the slightly acidic environment of the tumor, we linked doxorubicin to the targeted drug delivery system through a low pH-sensitive hydrazone bond. Effective release in the tumor microenvironment, thereby improving efficacy and reducing side effects. At the same time, due to the proton sponge effect of PEI, it can promote the lysosome escape of the drug and reduce the degradation of the drug in the lysosome, which can further improve the anti-tumor effect of the drug.

X-ray computed tomography (CT) imaging technology uses the difference in the penetration ability of X-rays to different substances. When it is absorbed, its degree of absorption is different, so images with different black and white intensities appear on the X-ray film. In tumor imaging technology, X-ray/CT imaging is currently the most commonly used diagnostic tool in hospitals, which can track the location, size and spread of tumors. However, currently used CT contrast agents are mainly iodine-containing (Z = 53) molecules (such as iodhirase, iopamidol, and iopromide, etc.), and these contrast agents are gradually becoming insufficient in clinical practice. For example, it is easy to be cleared by the kidneys so that the contrast time is very short, which is nephrotoxic. Moreover, X-rays can induce iodine-containing substances to ionize iodide ions, causing toxicity. However, the size of nanoparticles is small (1-100 nm), and they can enter micron-sized capillaries, so they can enter tissues more; moreover, due to the enhanced permeability of capillaries at cancerous tissue sites, nanoparticles can pass through Leakage deposits more on cancerous tissue, allowing better imaging of cancerous areas. At the same time, compared with iodine, gold has a higher atomic number (Au: 79; I: 53) and X-ray absorption coefficient (Au: 5.16 cm2/g at 100keV; I: 1.94 cm2/g), gold can significantly hinder Propagation of X-rays. Therefore, gold nanoparticles can be used in the development of CT contrast agents, and as X-ray imaging agents, they are expected to be used in the early diagnosis of tumors.

Nano-therapeutics integrates drugs and imaging reagents into nanoparticles, and delivers them to diseased tissues in a targeted manner, improving treatment effects and reducing side effects on normal tissues, and realizing real-time detection of tumor treatment effects in tumor treatment. It is of great significance to improve the treatment plan, improve the cure rate of tumor and relieve the suffering of patients. Among them, gold nanostars are expected to be used in the diagnosis and treatment of cancer due to their unique surface effect and size effect. Construct stable, non-toxic and biocompatible nanocarriers by optimizing carrier materials, use nanocarriers to combine anticancer drugs and targeting molecules, and integrate functions such as targeted drug delivery, live body real-time tracking, drug treatment and prognosis monitoring. The multifunctional nanosystems will be the research trend in the future, which will provide strong support for effectively improving drug delivery efficiency and reducing drug side effects. Gold nanostars will be able to realize the integration of cancer diagnosis and treatment, and have significant advantages such as strong pertinence, high treatment efficiency, safety and non-toxicity, bringing new opportunities for the integration of cancer diagnosis and treatment. So how to prepare multifunctional anti-tumor targeted diagnosis and treatment drugs based on gold nanostars has not been reported with practical and effective methods so far.

Contents of the invention

In view of the above situation, in order to overcome the defects in the prior art, the purpose of the present invention is to provide a preparation method and application of a multifunctional anti-tumor targeted diagnosis and treatment drug based on gold nanostars, which can effectively solve the toxicity of existing anti-tumor drugs. Large side effects, poor biocompatibility, and poor targeting.

The technical solution solved by the present invention is: first, carry out structural modification to the gold nanostar carrier, and react with an amino compound containing a sulfhydryl group to obtain an amino-modified gold nanostar solution; Carry out an amide reaction to generate a gold nanostar solution modified with a hydrazine bond at the end; then use the hydrazine bond at the end to chemically react with doxorubicin containing a carbonyl group to form a pH-sensitive hydrazone bond compound; finally, the compound A gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment drug (system) can be obtained through the reaction of thiojinjian and thiolated NGR. The specific steps are as follows:

(1) Preparation of aminated gold nanostars: Add a weight concentration of 0.1 to 10.0 mg/mL amino compound 1-5mL, stirred at room temperature in the dark for 6-12h, centrifuged at 10,000 rpm for 30min, and redissolved with ultrapure water to obtain an aminated gold nanostar solution;

The amino compound is NH ₂ -PEG2000-SH or PEI-SH;

(2) Preparation of gold nanostar solution modified by hydrazine bond: first weigh 1.5-4.5 mg of p-carboxyphenylhydrazine, dissolve in ultrapure water to obtain a carboxylation solution, and then weigh 1-(3-dimethylaminopropyl) Add 2.5-6.0 mg of -3-ethylcarbodiimide hydrochloride, 0.5-3.5 mg of N-hydroxysuccinimide and 1.6-2.0 mg of 4-dimethylaminopyridine into 1-3 mL of dimethyl sulfoxide in sequence , and then adding the carboxylation solution into the dimethyl sulfoxide solvent for carboxylation reaction to obtain an activation solution, adding the activation solution to the aminated gold nanostar solution prepared in step (1), under nitrogen protection, at room temperature to avoid Stir lightly for 24-48 hours, then centrifuge at 10,000 rpm for 30 minutes to remove free small molecules, and obtain a gold nanostar solution modified with hydrazine bonds;

(3) Preparation of doxorubicin-loaded gold nanostars: dry the hydrazine bond-modified gold nanostars solution prepared in step (2) to obtain a powder (GNSTs-PEG/PEI-HBA), mix the powder with doxorubicin (DOX ) were sequentially added to 10-20mL of reaction solvent at a weight ratio of 1:1.5-3.0, and reacted at room temperature in the dark for 24-48 hours to obtain a hydrazone bond-linked gold nanostar-borne anti-tumor drug solution, which was then centrifuged at 10,000 rpm for 30 minutes at high speed (to remove free doxorubicin and dimethyl sulfoxide), dried to obtain gold nanostars loaded with doxorubicin;

The drying is one of freeze-drying, vacuum drying or drying; the reaction solvent is one of dimethyl sulfoxide, dimethylformamide and phosphate buffer (neutral);

(4) Preparation of multifunctional anti-tumor targeted diagnosis and treatment drugs based on gold nanostars: put the doxorubicin-loaded gold nanostars prepared in step (3) under magnetic stirring at 18-25°C and 300-720 r/min Dissolve in 10-20 mL of ultra-pure water, then add 1-3 mg of NGR under nitrogen protection, and react at 25-35 °C, 200-400 r/min in the dark for 24-48 hours to finally obtain a multifunctional anti-tumor target based on gold nanostars Drugs for diagnosis and treatment.

The particle size of the gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment drug prepared by the present invention is 80-150nm, which can be effectively used in the preparation of active targeted anti-tumor drugs, or in the preparation of anti-tumor photodynamic heat-sensitive agents irradiated by radio frequency or in the preparation of anti-tumor early diagnosis X-ray imaging contrast agents, or in the preparation of anti-tumor delivery system drugs integrating diagnosis and treatment.

The multifunctional anti-tumor targeted diagnosis and treatment drug based on gold nanostars of the present invention can be loaded with chemotherapeutic drugs. Gold nanostars have the characteristics of generating hyperthermia and X-ray imaging under radio frequency irradiation. By utilizing the high temperature (>42°C) generated under radio frequency irradiation, it can promote the targeted release of doxorubicin at the tumor site to kill tumor cells , and gold nanostars can be used as contrast agents for X-Ray imaging. Thereby realizing the combined anti-tumor effect of diagnosis, hyperthermia and chemotherapy; the body of the present invention has good physical and chemical stability, simple preparation process, abundant sources of raw materials, low cost, little toxic and side effects, can effectively inhibit tumor cell proliferation, and is an anti-tumor targeting drug. Innovations in diagnostic and therapeutic drugs (carriers).

detailed description

The specific implementation of the present invention will be described in detail below in conjunction with the examples.

The present invention can be provided by the following examples in concrete implementation.

Example 1

In concrete implementation, the present invention can be realized by the following steps:

(1) Preparation of aminated gold nanostars: Add NH ₂ -PEG2000 with a weight concentration of 0.5 mg/mL to 20 mL of a gold nanostar solution with a weight concentration of 50 μg/mL at 25°C under magnetic stirring at 540 r/min 1mL of -SH solution and 1mL of PEI-SH solution with a weight concentration of 10/mg/mL were stirred at room temperature for 12h in the dark, centrifuged at 10,000 rpm for 30min to obtain a precipitate, and the precipitate was redissolved in 22mL of ultrapure water to obtain aminated gold nanoparticles Star (GNSTs-PEG/PEI) solution;

(2) Preparation of gold nanostar solution modified by hydrazine bond: first take 2.54 mg of p-carboxyphenylhydrazine, dissolve it in 0.2 mL of ultrapure water to obtain a carboxylation solution, and then weigh 1-(3-dimethylaminopropyl)- 4.06 mg of 3-ethylcarbodiimide hydrochloride, 1.78 mg of N-hydroxysuccinimide and 1.8 mg of 4-dimethylaminopyridine were sequentially added to 2 mL of dimethyl sulfoxide, and then the carboxylation solution was added to di Perform carboxylation reaction in methyl sulfoxide solvent to obtain an activation solution, add the activation solution to the aminated gold nanostar solution prepared in step (1), under nitrogen protection, stir and react at room temperature in the dark for 48 hours, and then centrifuge at 10000rpm at high speed After 30 minutes, free small molecules were removed to obtain a hydrazine bond-modified gold nanostar (GNSTs-PEG/PEI-HBA) solution;

(3) Preparation of doxorubicin-loaded gold nanostars: lyophilize the hydrazine bond-modified gold nanostar solution prepared in step (2) to obtain hydrazine bond-modified gold nanostars (GNSTs-PEG/PEI-HBA) powder, Add the powder and doxorubicin (DOX) to 20mL of dimethyl sulfoxide sequentially at a weight ratio of 1:2, and react in the dark for 48 hours at room temperature to obtain a hydrazone bond-linked gold nanostar-loaded anti-tumor drug solution, and then 10,000 rpm Centrifuge at high speed for 30 minutes (to remove free doxorubicin and dimethyl sulfoxide), freeze-dry to obtain gold nanostars loaded with doxorubicin (GNSTs-PEG/PEI-HBA-DOX);

(4) Preparation of multifunctional anti-tumor targeted diagnosis and treatment drugs based on gold nanostars: the doxorubicin-loaded gold nanostars prepared in step (3) were mixed with ultrapure water at 25°C under magnetic stirring at 540 r/min. 20mL was dissolved, and then NGR 2mg was added under the protection of nitrogen, and reacted in the dark at 35 ℃, 300r/min for 24h, and finally a multifunctional anti-tumor targeted diagnosis and treatment drug based on gold nanostars (NGR/GNSTs-PEG/PEI-HBA -dox).

Example 2

In specific implementation, the present invention can also be realized by the following steps:

(1) Preparation of aminated gold nanostars: Add NH ₂ -PEG2000-SH with a weight concentration of 5 mg/mL to 15 mL of a gold nanostar solution with a weight concentration of 50 μg/mL under magnetic stirring at 19°C and 700 r/min 3mL, stirred at room temperature in the dark for 9h, centrifuged at 10,000 rpm for 30min at high speed, and redissolved in ultrapure water to obtain aminated gold nanostar (GNSTs-PEG) solution;

(2) Preparation of gold nanostar solution modified with hydrazine bond: first weigh 3 mg of p-carboxyphenylhydrazine, dissolve it in ultrapure water to obtain a carboxylation solution, and then weigh 1-(3-dimethylaminopropyl)-3- Add 4.5 mg of ethylcarbodiimide hydrochloride, 2 mg of N-hydroxysuccinimide and 1.7 mg of 4-dimethylaminopyridine to 2 mL of dimethyl sulfoxide in sequence, and then add the carboxylation solution to dimethyl sulfoxide The carboxylation reaction was carried out in a sulfone solvent to obtain an activation solution, and the activation solution was added to the aminated gold nanostar solution prepared in step (1), under the protection of nitrogen, stirred at room temperature and protected from light for 36 hours, and then centrifuged at 10,000 rpm for 30 minutes at high speed, Remove free small molecules to obtain a hydrazine bond-modified gold nanostar (GNSTs-PEG-HBA) solution;

(3) Preparation of doxorubicin-loaded gold nanostars: vacuum-dry the hydrazine bond-modified gold nanostars solution prepared in step (2) to obtain a powder (GNSTs-PEG-HBA), mix the powder with doxorubicin (DOX) Add it to 15mL of dimethylformamide in sequence at a weight ratio of 1:1.8, and react in the dark for 36 hours at room temperature to obtain a hydrazone bond-linked gold nanostar-borne anti-tumor drug solution, and then centrifuge at 10,000 rpm for 30 minutes at a high speed (to remove free doxorubicin). and dimethyl sulfoxide), vacuum-dried to obtain doxorubicin-loaded gold nanostars (GNSTs-PEG-HBA-DOX);

(4) Preparation of multifunctional anti-tumor targeted diagnosis and treatment drugs based on gold nanostars: the doxorubicin-loaded gold nanostars prepared in step (3) were mixed with 15 mL of ultrapure water at 22 °C and 500 r/min magnetic stirring Dissolve, then add 2 mg of NGR under the protection of nitrogen, react at 30°C, 300r/min in the dark for 36 hours, and finally obtain a gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment drug (NGR/GNSTs-PEG-HBA-DOX) .

Example 3

In specific implementation, the present invention can also be realized by the following steps:

(1) Preparation of aminated gold nanostars: At 23°C, under magnetic stirring at 400 r/min, add 4 mL of PEI-SH with a weight concentration of 9 mg/mL to 18 mL of a gold nanostar solution with a weight concentration of 50 μg/mL, and store at room temperature Stir and react in the dark for 10 hours, centrifuge at 10,000 rpm for 30 minutes at high speed, and redissolve with ultrapure water to obtain aminated gold nanostar (GNSTs-PEI) solution;

(2) Preparation of gold nanostar solution modified with hydrazine bond: first weigh 3.5 mg of p-carboxyphenylhydrazine, dissolve it in ultrapure water to obtain a carboxylation solution, and then weigh 1-(3-dimethylaminopropyl)-3 -Ethylcarbodiimide hydrochloride 4mg, N-hydroxysuccinimide 3mg and 4-dimethylaminopyridine 1.9mg were sequentially added to 2.5mL dimethyl sulfoxide, and then the carboxylation solution was added to dimethyl Perform carboxylation reaction in a sulfoxide solvent to obtain an activation solution, add the activation solution to the aminated gold nanostar solution prepared in step (1), under the protection of nitrogen, stir and react at room temperature in the dark for 40 hours, and then centrifuge at 10,000 rpm for 30 minutes , to remove free small molecules to obtain a hydrazine bond-modified gold nanostar (GNSTs-PEI-HBA) solution;

(3) Preparation of doxorubicin-loaded gold nanostars: drying the hydrazine bond-modified gold nanostars solution prepared in step (2) to obtain a powder (GNSTs-PEI-HBA), and mixing the powder with doxorubicin (DOX) Added to 12mL reaction solvent in sequence at a weight ratio of 1:1.6, and reacted at room temperature in the dark for 40 hours to obtain a hydrazone bond-linked gold nanostar-borne anti-tumor drug solution, and then centrifuged at 10,000 rpm for 30 minutes at a high speed (to remove free doxorubicin and Methyl sulfoxide), dried to obtain gold nanostars loaded with doxorubicin (GNSTs-PEI-HBA-DOX);

(4) Preparation of multifunctional anti-tumor targeted diagnosis and treatment drugs based on gold nanostars: the doxorubicin-loaded gold nanostars prepared in step (3) were mixed with 18 mL of ultrapure water at 23°C and 400 r/min under magnetic stirring. Dissolve, then add NGR 2.5mg under the protection of nitrogen, and react for 40 hours at 28°C, 350r/min in the dark, and finally get the multifunctional anti-tumor targeted diagnosis and treatment drug based on gold nanostars (NGR/GNSTs-PEI-HBA-DOX ).

Example 4

The present invention can also be realized by the following steps in concrete implementation:

(1) Preparation of aminated gold nanostars: at 20-22°C, under magnetic stirring at 500-600 r/min, add 4-6 mg/mL of gold nanostar solution with a weight concentration of 50 μg/mL 2-4 mL of amino compound, stirred at room temperature and protected from light for 8-10 hours, centrifuged at 10,000 rpm for 30 minutes, and redissolved with ultrapure water to obtain an aminated gold nanostar solution;

(2) Preparation of gold nanostar solution modified by hydrazine bond: first weigh 2.5-3.5 mg of p-carboxyphenylhydrazine, dissolve in ultrapure water to obtain a carboxylation solution, and then weigh 1-(3-dimethylaminopropyl) Add 3-4mg of 3-ethylcarbodiimide hydrochloride, 1.5-2.5mg of N-hydroxysuccinimide and 1.7-1.9mg of 4-dimethylaminopyridine into 1.5-2.5mL dimethyl sulfoxide in sequence , and then adding the carboxylation solution into the dimethyl sulfoxide solvent for carboxylation reaction to obtain an activation solution, adding the activation solution to the aminated gold nanostar solution prepared in step (1), under nitrogen protection, at room temperature to avoid Stir lightly for 30-35 hours, then centrifuge at 10,000 rpm for 30 minutes to remove free small molecules, and obtain a solution of gold nanostars modified with hydrazine bonds;

(3) Preparation of doxorubicin-loaded gold nanostars: dry the hydrazine bond-modified gold nanostars solution prepared in step (2) to obtain a powder (GNSTs-PEG/PEI-HBA), mix the powder with doxorubicin (DOX ) was sequentially added to 14-16mL of reaction solvent at a weight ratio of 1:2-2.5, and reacted at room temperature in the dark for 30-35 hours to obtain a hydrazone bond-linked gold nanostar-borne anti-tumor drug solution, and then centrifuged at 10,000 rpm for 30 minutes at high speed (to remove free doxorubicin and dimethyl sulfoxide), dried to obtain gold nanostars loaded with doxorubicin;

(4) Preparation of multifunctional anti-tumor targeted diagnosis and treatment drugs based on gold nanostars: put the doxorubicin-loaded gold nanostars prepared in step (3) under magnetic stirring at 20-22°C and 500-600 r/min Dissolve in 14-16 mL of ultrapure water, then add 1.5-2.5 mg of NGR under nitrogen protection, and react for 30-40 hours at 28-32 ° C, 260-340 r/min in the dark, and finally obtain a multifunctional anti-tumor drug based on gold nanostars Targeted diagnostic therapy drugs.

The gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment drug prepared by the method of the present invention has a particle size of 80-150 nm, and is used in active targeting anti-tumor, and is used as a thermosensitizer in anti-tumor under radio frequency irradiation In terms of photodynamic therapy, as an X-ray imaging contrast agent in the early diagnosis of anti-tumor, as an anti-tumor drug delivery system integrating diagnosis and treatment. After testing, very good beneficial technical effects have been achieved. The application of the multifunctional anti-tumor targeted diagnosis and treatment system based on gold nanostars in anti-tumor treatment of the present invention is divided into two parts: in vitro and in vivo:

(1) In vitro: the multifunctional anti-tumor targeted diagnosis and treatment system based on gold nanostars of the present invention was added to tumor cells for cultivation, and radiofrequency irradiation (13.56MHz, 1-5 min) was used 4-6 hours after administration, Then change to a fresh medium and continue to incubate for 24 hours to measure the survival rate of tumor cells.

The above-mentioned cancer cells are: various solid tumors that appear on the surface or inside of organs, lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, leukemia, pancreatic cancer, cervical cancer, Laryngeal cancer, thyroid cancer, tongue cancer, brain tumor (intracranial tumor), small intestine tumor, gallbladder cancer, bile duct cancer, kidney cancer, prostate cancer, penile cancer, Cuiwan tumor, endometrial cancer, choriocarcinoma, vaginal malignancy Tumor, malignancy of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, malignant melanoma.

(2) In vivo: the multifunctional anti-tumor targeted diagnosis and treatment system based on gold nanostars of the present invention was intravenously injected into the tumor-bearing mice, and radiofrequency irradiation (13.56 MHz, 1-5 min) was used 4 to 6 hours after administration, and the radiation Finally, the temperature of the irradiated part was recorded with an infrared thermal camera.

The tumor-bearing mice mentioned above are: various solid tumors appearing on the surface or inside of organs, lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, leukemia, pancreatic cancer, cervical cancer Cancer, laryngeal cancer, thyroid cancer, tongue cancer, brain tumor (intracranial tumor), small intestine tumor, gallbladder cancer, bile duct cancer, kidney cancer, prostate cancer, penile cancer, Tsui Wan tumor, endometrial cancer, choriocarcinoma, One of vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, and malignant melanoma.

The novel gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system of the present invention can be made into various pharmaceutical dosage forms as a heat-sensitive agent, such as injections, sterile powder injections for injections, dispersions, patches, gels, Implants etc. Various preparation additives can be added to the preparation of the present invention, such as physiological saline, glucose, buffer solution and preservatives and the like. The administration method can be intravenous injection, intramuscular injection, intratumoral injection and subcutaneous injection, transdermal administration, internal implantation and the like.

The relevant test data are as follows:

1. The present invention is based on the determination of the drug loading of the gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system:

Taking the multifunctional anti-tumor targeted diagnosis and treatment system based on gold nanostars of the present invention, its drug loading is determined to be 1.16 mg/mL by ultraviolet spectrophotometry, indicating that the multifunctional anti-tumor targeted diagnosis and treatment based on gold nanostars of the present invention The system can be used as a carrier of antitumor drugs.

2. The present invention is based on the determination of the particle size and surface charge of the multifunctional anti-tumor targeted diagnosis and treatment system based on gold nanostars:

In the present invention, the particle size and surface charge of the multifunctional anti-tumor targeted diagnosis and treatment system based on gold nanostars are measured using a Nano-ZS90 laser particle size analyzer. The refractive index is set to 1.590 and the absorption coefficient is set to 0.010. , the temperature is set to 25°C, the measurement mode is set to automatic, and the Z average statistical value is used as the measurement result. Three copies of each horizontal condensate were prepared, each was measured once, and the average value of the three measurements was taken as the measurement result. The dielectric constant is set to 79, the viscosity coefficient is set to 0.8872, the temperature is set to 25°C, and the measurement mode is set to automatic. Three copies of each horizontal condensate were prepared, each was measured once, and the average value of the three measurements was taken as the measurement result. The measured results showed that the particle size was 80-150nm, and the potential was +10-30mV.

3. Using light irradiation to measure the growth activity of tumor cells by the gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system of the present invention:

In vitro anti-tumor activity experiment of a multifunctional anti-tumor targeted diagnosis and treatment system based on gold nanostars by radiofrequency irradiation: MCF-7 breast cancer cells (provided by Shanghai Cell Bank) were used as cancer cells to be investigated. Human breast cancer cells MCF-7 were cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% double antibody, and incubated in a cell culture incubator at 37°C containing 5% CO ₂ , and the medium was changed every other day. Digest and passage once in 2 to 3 days with 0.25% trypsin (containing 0.02% EDTA), and perform relevant experiments when the cells grow to 70% to 80%. Cells in logarithmic growth phase were used in all experiments. The SRB method was used to detect the toxic effect of gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system on MCF-7 cells. Take the cells in the logarithmic growth phase, routinely digest them into a single-cell suspension, and count them. Adjust the density of the cells to be tested to 5×10 ³ cells/well (the edge wells are filled with sterile PBS), spread 96-well plates, and add 200 μl of culture to each well. cultured in a cell incubator at 37°C with 5% CO ₂ . After 24 hours when the cells were all attached to the wall, the original medium was discarded, and the experimental group was added the multifunctional anti-tumor target based on gold nanostars diluted in the medium with a concentration gradient of (0.5, 1, 5, 10, 50, 100 μg/mL). For the diagnosis and treatment system, set 3 to 5 duplicate wells for each concentration, add blank culture medium to the blank cell group, set 3 to 5 duplicate wells, as the control group, and continue to culture in the incubator. 4 hours after the addition of drugs in the radio frequency group, use radio frequency irradiation (13.56MHz, 2min). After the light is over, place the cell culture plate in the incubator to continue culturing for 24 hours, then stop the culture, and gently add 25 μl of pre-cooled 50% TCA was fixed, and after standing at room temperature for 5 minutes, the cell plate was moved to a refrigerator at 4°C for 1 hour, so that the suspended cells could be fixed at the bottom of the culture well. Pour off the fixative, wash each duplicate well 5 times with deionized water, air dry, and dry completely. Add 50 μl of 0.4% SRB staining solution prepared with 1% acetic acid to each well, keep away from light for 20-30 minutes at room temperature, discard the staining solution, and wash 5 times with 1% acetic acid to remove unbound dye. After air drying, dissolve with 10mmol/L (pH10.5) 150μl/well unbuffered Tris base. After standing at room temperature for 5 minutes, place the cell plate on a shaker for 5 minutes, which will improve the mixing of the dye. An automatic microplate reader can be used to measure the absorbance of the control group and the experimental group respectively at the wavelengths of 565nm and 690nm. Calculate the cell inhibition rate of each concentration according to the formula: cell inhibition rate=1-(experimental group OD value/control group OD value)×100%.

The experimental results show that the gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system of the present invention enters the interior of tumor cells, exerts good curative effect of anti-tumor drugs, and can inhibit tumor cell proliferation more obviously after combined with radio frequency irradiation.

Four, the determination of the in vivo antitumor activity of the multifunctional antitumor targeted diagnosis and treatment system based on gold nanostars in the present invention:

MCF-7 cells were inoculated subcutaneously under the right anterior axilla of BALB/c nude mice at 1×10 ⁷ cells/mouse. Seven days after tumor inoculation in nude mice, 36 nude mice with tumor volume ≥ 100 ^mm3 were randomly divided into 6 groups, 6 mice in each group. The specific groups are as follows: (1) control group (normal saline group); (2) normal saline radiofrequency group; (3) targeted drug group; (4) targeted drug radiofrequency group; (5) non-targeted drug group; 6) Non-targeted drug radio frequency group. The 6 groups all adopted tail vein administration, and the radiofrequency group irradiated the tumor site 4 hours after administration, using the most commonly used radiofrequency frequency of 13.56MHz (1min). Administer once every other day, inject 200 μl of physiological saline or drug solution or 100 μg/mL preparation solution each time, and administer 3 times in total. During the whole experiment, the living conditions of the nude mice were observed daily, their body weight was weighed every other day, and the long diameter (A) and short diameter (B) of the nude mouse tumors were measured with a vernier caliper, according to the formula V=1/2 (A×B ² ) to calculate the tumor volume.

Experiments have proved that when the gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system of the present invention is administered, the increase in tumor volume in nude mice is significantly inhibited compared with the single targeting group or the single radiofrequency group. After combined with radiofrequency irradiation, The increase of tumor volume in nude mice can be more significantly inhibited.

Five, the present invention is based on gold nanostar multifunctional anti-tumor targeted diagnosis and treatment system as X-ray imaging agent in the early diagnosis of tumor application:

The 12 mice inoculated with MCF-7 breast cancer tumor cells were randomly divided into experimental group and control group, and 6 mice in the experimental group were injected with 200μl 1.16mg/mL NGR/GNSTs-PEG/PEI-HBA through the tail vein -DOX solution. The other 6 mice in the control group were not given any treatment. Mice were anesthetized by intraperitoneal injection of 3% pentobarbital sodium 0.04mL, and before injection of NGR/GNSTs-PEG/PEI-HBA-DOX solution (ie 0 h) and injection of NGR/GNSTs-PEG/PEI-HBA -X-ray imaging was carried out 24h after DOX. On the T2WI images of the tumor areas of mice in the experimental group and the control group at 0h and 24h, a region of interest (ROI) of the same size was drawn to measure the signal intensity of the solid part of the tumor (SI _T ), and the average value was taken. The measurement area was not less than 6mm ² . Using the SPSS 19.0 statistical software package, the measurement data conforming to the normal distribution is expressed as x±S, and the discrepancy is expressed as the median (M). Two independent sample t tests were used to compare the signal intensity of tumor solid part (SIT) between the experimental group and the control group at 0h and 24h. P<0.05 means the difference is statistically significant, and p<0.01 means the difference is highly statistically significant.

It has been confirmed that the gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system of the present invention can be used as an X-ray imaging contrast agent for early diagnosis of tumors.

The gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system provided by the present invention is expected to be a good heat-sensitive agent for treating tumors, and can be used as a carrier for chemical drugs, proteins, and nucleic acids, and can also be used as an X- The ray imaging contrast agent is a great innovation in drug preparation.

Compared with the prior art, the present invention has the following outstanding beneficial technical effects:

(1) The new gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system of the present invention will not destroy the characteristics of the gold nanostar itself, has good water dispersibility, and has low toxicity to organisms. Physical and chemical It has good stability, can be placed in an aqueous solution at 4°C for three months, its particle size does not change significantly, the preparation conditions are easy to meet, and the source of raw materials is abundant.

(2) The gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system provided by the present invention, as a good thermosensitive agent in anti-tumor thermosensitive therapy, can exert anti-tumor activity by generating heat energy during radio frequency irradiation, However, when radio frequency irradiation is not used, the antitumor activity of the drug delivery system is very small.

(3) The gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system provided by the present invention, as a pH-sensitive drug delivery system, can be phagocytized by cells when the pH is less than or equal to 6.4, and can be phagocytized by cells when the pH is greater than or equal to When 7.4, it is not easy to be phagocytized by cells. This property can be used to identify the characteristic slightly acidic environment of tumors. At the same time, the drug system is targeted and can produce overheating phenomenon under radio frequency irradiation, which can not only improve the anti-tumor curative effect, the curative effect of the combined treatment group is 1.6 times higher than that of the drug group alone, but also can significantly reduce its damage to surrounding tissues. toxic side effects.

(4) The gold nanostar-based multifunctional anti-tumor targeted diagnosis and treatment system provided by the present invention, as a good X-ray imaging contrast agent, has a good negative enhancement effect and good tumor imaging effect. -ray imaging can monitor the effect of tumor treatment in real time, which has strong practicability and huge economic and social benefits.

Claims (3)

1. a kind of preparation method of the multi-functional antineoplastic target diagnoses and treatment medicine based on gold nano star, it is characterised in that first Structural modification first is carried out to the spaceborne body of gold nano, is reacted with the amino-compound containing sulfydryl, obtains amido modified gold nano Star solution；The hydrazine key compound of amido modified gold nano star and activated carboxyl is subjected to acid amides reaction, the connection of generation end again There is the gold nano star solution that hydrazine key is modified；Then the hydrazine key of its end is recycled to carry out chemistry with the adriamycin containing carbonyl anti- Should, formed with hydrazone key compound sensitive pH；Finally, this compound is reacted by the NGR of sulphur Jinjian and sulfhydrylation, produced Multi-functional antineoplastic target diagnoses and treatment medicine based on gold nano star, is comprised the following steps that：

（1）Prepare amidized gold nano star：Under 18~25 DEG C, 300~720r/min magnetic agitation, it is in weight concentration Added in the 50 μ g/mL mL of gold nano star solution 10~20 weight concentration 0.1~10.0 mg/mL amino-compound 1~ 5mL, room temperature lucifuge stirring reaction 6~12h, 10000 rpm high speed centrifugation 30min, then redissolved with ultra-pure water, obtain amidized gold Nanometer star solution；

Described amino-compound is PEI-SH；

（2）Prepare the gold nano star solution of hydrazine key modification：First weigh to the mg of carboxyl phenylhydrazine 1.5~4.5, dissolved, obtained with ultra-pure water Carboxylated solution, then weigh 1-（3- dimethylamino-propyls）- 3- ethyl-carbodiimide hydrochlorides 2.5~6.0 mg, N- hydroxyl amber The mg of amber acid imide 0.5~3.5 and DMAP 1.6-2.0mg are sequentially added in 1~3mL dimethyl sulfoxide (DMSO)s, then will Carboxylated solution, which is added in dimethyl sulfoxide solvent, carries out carboxylation reaction, obtains activated solution, activated solution is added into step （1）In the amidized gold nano star solution prepared, under nitrogen protection, room temperature lucifuge 24~48h of stirring reaction, then The min of 10000rpm high speed centrifugations 30, removes free small molecule, obtains the gold nano star solution of hydrazine key modification；

（3）Prepare the gold nano star for carrying adriamycin：By step（2）The gold nano star solution of the hydrazine key modification of preparation is dried, and obtains powder End, by powder and adriamycin with weight ratio 1：1.5~3.0 are sequentially added in 10~20 mL reaction dissolvent, the reaction of room temperature lucifuge 24~48h, is able to the spaceborne antineoplastic drug solution of gold nano of hydrazone key connection, then 10000 rpm high speed centrifugation 30min, dries, The gold nano star of adriamycin must be carried；

Described drying is one kind in being freeze-dried, be dried in vacuo or drying；Reaction dissolvent is dimethyl sulfoxide (DMSO), dimethyl methyl One kind in acid amides and phosphate buffer；

（4）Prepare the multi-functional antineoplastic target diagnoses and treatment medicine based on gold nano star：By step（3）The load adriamycin of preparation Gold nano star dissolved under 18~25 DEG C, 300~720 r/min magnetic agitation with 10~20mL of ultra-pure water, then in nitrogen Under protection, add NGR 1~3mg, 25~35 DEG C, 200~400r/min lucifuges react 24~48h, it is final to be based on Jenner Meter Xing multi-functional antineoplastic target diagnoses and treatment medicine.

2. the preparation side of the multi-functional antineoplastic target diagnoses and treatment medicine according to claim 1 based on gold nano star Method, it is characterised in that realized by following steps：

（1）Prepare amidized gold nano star：It is 50 μ g/mL's in weight concentration under 23 DEG C, 400r/min magnetic agitation Add weight concentration 9mg/mL PEI-SH 4mL in gold nano star solution 18mL, room temperature lucifuge stirring reaction 10h, 10000 Rpm high speed centrifugation 30min, then redissolved with ultra-pure water, obtain amidized gold nano star solution；

（2）Prepare the gold nano star solution of hydrazine key modification：First weigh to carboxyl phenylhydrazine 3.5mg, dissolved with ultra-pure water, obtain carboxylated Solution, then weigh 1-（3- dimethylamino-propyls）- 3- ethyl-carbodiimide hydrochlorides 4mg, n-hydroxysuccinimide 3mg and 4- Dimethylamino naphthyridine 1.9mg is sequentially added in 2.5mL dimethyl sulfoxide (DMSO)s, and it is molten that carboxylated solution then is added into dimethyl sulfoxide (DMSO) Carboxylation reaction is carried out in agent, activated solution is obtained, activated solution is added into step（1）The amidized gold nano star solution prepared In, under nitrogen protection, room temperature lucifuge stirring reaction 40h, then the min of 10000rpm high speed centrifugations 30, remove free small point Son, obtains the gold nano star solution of hydrazine key modification；

（3）Prepare the gold nano star for carrying adriamycin：By step（2）The gold nano star solution drying of the hydrazine key modification of preparation, obtains powder End, by powder and adriamycin with weight ratio 1：1.6 are sequentially added in 12mL reaction dissolvent, room temperature lucifuge reaction 40h, are able to hydrazone The spaceborne antineoplastic drug solution of gold nano of key connection, then 10000 rpm high speed centrifugation 30min, drying are dry, must carry adriamycin Gold nano star；

（4）Prepare the multi-functional antineoplastic target diagnoses and treatment medicine based on gold nano star：By step（3）The load adriamycin of preparation Gold nano star dissolved under 23 DEG C, 400r/min magnetic agitation with ultra-pure water 18mL, then under nitrogen protection, add NGR 2.5mg, 28 DEG C, 350r/min lucifuges reaction 40h, it is final to obtain the multi-functional antineoplastic target diagnoses and treatment based on gold nano star Medicine.

3. the preparation side of the multi-functional antineoplastic target diagnoses and treatment medicine according to claim 1 based on gold nano star Method, it is characterised in that realized by following steps：

（1）Prepare amidized gold nano star：Under 20~22 DEG C, 500~600r/min magnetic agitation, it is in weight concentration 4~6mg/mL of weight concentration 2~4mL of amino-compound is added in 50 μ g/mL 14~16mL of gold nano star solution, room temperature is kept away Light stirring reaction 8~10h, 10000 rpm high speed centrifugation 30min, then redissolved with ultra-pure water, obtain amidized gold nano star molten Liquid；

（2）Prepare the gold nano star solution of hydrazine key modification：First weigh to 2.5~3.5mg of carboxyl phenylhydrazine, dissolved with ultra-pure water, obtained Carboxylated solution, then weigh 1-（3- dimethylamino-propyls）3~4mg of -3- ethyl-carbodiimide hydrochlorides, N- hydroxysuccinimidyls acyl are sub- 1.5~2.5mg of amine and 1.7~1.9mg of DMAP are sequentially added in 1.5~2.5mL dimethyl sulfoxide (DMSO)s, then by carboxylic Base solution, which is added in dimethyl sulfoxide solvent, carries out carboxylation reaction, obtains activated solution, activated solution is added into step（1） In the amidized gold nano star solution prepared, under nitrogen protection, room temperature lucifuge 30~35h of stirring reaction, then 10000rpm The min of high speed centrifugation 30, removes free small molecule, obtains the gold nano star solution of hydrazine key modification；

（3）Prepare the gold nano star for carrying adriamycin：By step（2）The gold nano star solution of the hydrazine key modification of preparation is dried, and obtains powder End, by powder and adriamycin with weight ratio 1：2~2.5 are sequentially added in 14~16mL reaction dissolvent, room temperature lucifuge reaction 30 ~35h, is able to the spaceborne antineoplastic drug solution of gold nano of hydrazone key connection, then 10000 rpm high speed centrifugation 30min, dries, obtain Carry the gold nano star of adriamycin；

（4）Prepare the multi-functional antineoplastic target diagnoses and treatment medicine based on gold nano star：By step（3）The load adriamycin of preparation Gold nano star dissolved under 20~22 DEG C, 500~600 r/min magnetic agitation with 14~16mL of ultra-pure water, then in nitrogen Under protection, add NGR 1.5~2.5mg, 28~32 DEG C, 260~340r/min lucifuges react 30~40h, it is final must be based on gold The multi-functional antineoplastic target diagnoses and treatment medicine of nanometer star.